A primary goal in HIV/AIDS vaccine development is to elicit broadly neutralizing antibodies (bnAbs) against the HIV-1 envelope glycoprotein spike that is anchored in the membrane (m-Env). To date, experimental vaccines have elicited mostly narrow, weak or non-neutralizing antibodies, typically using soluble Env (s-Env) molecules. Here, we will use membrane Env liposome (MEL) vaccines that incorporate well-ordered and stabilized m-Env trimers into liposomes via the transmembrane domain. MELs thus present a relevant conformation of Env in a multivalent manner and contain important epitopes of the membrane proximal external region (MPER) that are missing from s-Env vaccines. The creation of MELs has been made feasible by recent improvements to m-Env production by creation of high Env producer cells. Meanwhile, B cell anergy is common among HIV-1 bnAb lineages but is not well addressed by traditional vaccines. Preliminary data show initial promise of MELs in eliciting nAb responses in human CD4BS bnAb CH103 UCA heterozygous dKI (HC+LC KI) mice in which ?on-target? anergy prone B cells had responded poorly to s-Env vaccines. In Aim 1, we will build on these findings by using consensus Env MELs, and mixed Env MELs, combined with selective removal of N-glycan at position 197 to increase accessibility to the CD4 binding site in order to expedite elicitation of cross-neutralizing antibodies in the prime. This will be followed by sequential boosting with MELs. The ?Booster MELs? will be consensus Envs, or those selected in part based on specific binding by antecedent serum antibodies from prior immunization, with the intent to drive affinity maturation of B cells against conserved elements of the target site. In an effort to further broaden nAb responses, we will use an approach described recently that elicits bnAbs to the fusion peptide (FP) in multiple species including mice. In this approach, a prime-boost regimen in CH103 UCA het dKI mice consisting of an FP-KLH prime and MEL boosts is designed to ?co-elicit? CD4BS bnAbs and FP bnAbs. In Aim 2, we will test a similar sequential MEL immunization strategy designed to elicit MPER-directed bnAbs in 2F5 KI mice, whose B-cells are also under more significant anergy controls. Hence, we will use strong universal T helper cell epitopes as a strategy to further help break B cell anergy to maximize bnAb responses. This will involve ?pre-priming? with universal Th epitopes using a lentiviral vaccine vector (LVV). Finally, in Aim 3, down-selected m-Env vaccine candidates eliciting the best nAb responses, and ideally comprising practical regimens that elicit nAbs to CD4BS, FP and MPER with the least boosts, will be used to immunize in fully polyclonal systems, i.e. rabbits and humanized Ig locus KI (Trianni) mice. The creation of m-Env-based vaccine schemes able to elicit cross-neutralizing antibodies against key vaccine targets, particularly in humanized polyclonal Ig KI mice, should have a significant impact on HIV-1 vaccine development.